Julien Lancelot

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.

Schistosoma mansoni Sirtuins: Characterization and Potential as Chemotherapeutic Targets

Background The chemotherapy of schistosomiasis currently depends on the use of a single drug, praziquantel. In order to develop novel chemotherapeutic agents we are investigating enzymes involved in the epigenetic modification of chromatin. Sirtuins are NAD+ dependent lysine deacetylases that are involved in a wide variety of cellular processes including histone deacetylation, and have been demonstrated to be therapeutic targets in various pathologies, including cancer. Methodology, Principal Findings In order to determine whether Schistosoma mansoni sirtuins are potential therapeutic targets we first identified and characterized their protein sequences. Five sirtuins (SmSirt) are encoded in the S. mansoni genome and phylogenetic analysis showed that they are orthologues of mammalian Sirt1, Sirt2, Sirt5, Sirt6 and Sirt7. Both SmSirt1 and SmSirt7 have large insertion in the catalytic domain compared to their mammalian orthologues. SmSirt5 is the only mitochondrial sirtuin encoded in the parasite genome (orthologues of Sirt3 and Sirt4 are absent) and transcripts corresponding to at least five splicing isoforms were identified. All five sirtuins are expressed throughout the parasite life-cycle, but with distinct patterns of expression. Sirtuin inhibitors were used to treat both schistosomula and adult worms maintained in culture. Three inhibitors in particular, Sirtinol, Salermide and MS3 induced apoptosis and death of schistosomula, the separation of adult worm pairs, and a reduction in egg laying. Moreover, Salermide treatment led to a marked disruption of the morphology of ovaries and testes. Transcriptional knockdown of SmSirt1 by RNA interference in adult worms led to morphological changes in the ovaries characterized by a marked increase in mature oocytes, reiterating the effects of sirtuin inhibitors and suggesting that SmSirt1 is their principal target. Conclusion, Significance Our data demonstrate the potential of schistosome sirtuins as therapeutic targets and validate screening for selective sirtuin inhibitors as a strategy for developing new drugs against schistosomiasis.

Structure-Based Design and Synthesis of Novel Inhibitors Targeting HDAC8 from Schistosoma mansoni for the Treatment of Schistosomiasis

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, a series of new benzohydroxamates were prepared as potent inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by these 3-amidobenzohydroxamates. The newly designed inhibitors were evaluated in screens for enzyme inhibitory activity against schistosome and human HDACs. Twenty-seven compounds were found to be active in the nanomolar range, and some of them showed selectivity toward smHDAC8 over the major human HDACs (1 and 6). The active benzohydroxamates were additionally screened for lethality against the schistosome larval stage using a fluorescence-based assay. Four of these showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture.

Molecular Basis for the Antiparasitic Activity of a Mercaptoacetamide Derivative That Inhibits Histone Deacetylase 8 (HDAC8) from the Human Pathogen Schistosoma mansoni

Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion and the essential catalytic tyrosine (Y341) residue via its mercaptoacetamide warhead. To our knowledge, this is the first complex crystal structure of any HDAC inhibited by a mercaptoacetamide inhibitor, and therefore, this finding offers a rationale for further improvement. Finally, an ester prodrug of the thiol HDAC inhibitor exhibited antiparasitic activity on cultured schistosomes in a dose-dependent manner.

Dual targeting of insulin and venus kinase Receptors of Schistosoma mansoni for novel anti-schistosome therapy.

Background Chemotherapy of schistosomiasis relies on a single drug, Praziquantel (PZQ) and mass-use of this compound has led to emergence of resistant strains of Schistosoma mansoni, therefore pointing out the necessity to find alternative drugs. Through their essential functions in development and metabolism, receptor tyrosine kinases (RTK) could represent valuable drug targets for novel anti-schistosome chemotherapies. Taking advantage of the similarity between the catalytic domains of S. mansoni insulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studied the possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug. Methodology/Principal Findings Several commercial RTK inhibitors were tested for their potential to inhibit the kinase activities of SmIR1, SmIR2, SmVKR1 and SmVKR2 intracellular domains (ICD) expressed in Xenopus oocytes. We measured the inhibitory effect of chemicals on meiosis resumption induced by the active ICD of the schistosome kinases in oocytes. The IR inhibitor, tyrphostin AG1024, was the most potent inhibitory compound towards SmIR and SmVKR kinases. In vitro studies then allowed us to show that AG1024 affected the viability of both schistosomula and adult worms of S. mansoni. At micromolar doses, AG1024 induced apoptosis and caused schistosomula death in a dose-dependent manner. In adult worms, AG1024 provoked alterations of reproductive organs, as observed by confocal laser scanner microscopy. With 5 µM AG1024, parasites were no more feeding and laying eggs, and they died within 48 h with 10 µM. Conclusion/Significance IRs and VKRs are essential in S. mansoni for key biological processes including glucose uptake, metabolism and reproduction. Our results demonstrate that inhibiting the kinase potential and function of these receptors by a single chemical compound AG1024 at low concentrations, leads to death of schistosomula and adult worms. Thus, AG1024 represents a valuable hit compound for further design of anti-kinase drugs applicable to anti-schistosome chemotherapy.

Targeting schistosome histone modifying enzymes for drug development.

The histone modifying enzymes (HME) represent particularly promising targets for the development of alternatives to praziquantel, the only currently available drug to combat schistosomiasis. The inhibition of these enzymes frequently arrests the cell cycle or induces apoptosis in cancer cells, but not in normal cells and numerous HME inhibitors are under investigation as potential anticancer agents. The recent resolution of the genome sequences of Schistosoma mansoni and Schistosoma japonicum has allowed us to identify all the schistosome genes encoding histone acetyltransferases, deacetylases, methyltransferases and demethylases. We have chosen a strategy using phylogenetic screening with inhibitors of HME classes, screening of individual HME targets by both high-throughput and reasoned (in silico docking using resolved crystal structures) approaches in a project funded by the European Community, named SEtTReND (Schistosome Epigenetics: Targets, Regulation, New Drugs). The initial focus is on the class I histone deacetylase (HDAC) 8 since the comparison of the catalytic site of the schistosome and human enzymes shows crucial differences, rendering possible the development of inhibitors specific for SmHDAC8. However, phenotypic screening shows that inhibitors of all HME classes tested were able to induce apoptosis and death in parasites in vitro, indicating that other enzymes may prove to be viable targets.

Epigenetic Changes Modulate Schistosome Egg Formation and Are a Novel Target for Reducing Transmission of Schistosomiasis

Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Epigenetic control of gene function in schistosomes: a source of therapeutic targets?

The discovery of the epigenetic regulation of gene expression has revolutionized both our understanding of how genomes function and approaches to the therapy of numerous pathologies. Schistosomes are metazoan parasites and as such utilize most, if not all the epigenetic mechanisms in play in their vertebrate hosts: histone variants, histone tail modifications, non-coding RNA and, perhaps, DNA methylation. Moreover, we are acquiring an increasing understanding of the ways in which these mechanisms come into play during the complex schistosome developmental program. In turn, interest in the actors involved in epigenetic mechanisms, particularly the enzymes that carry out epigenetic modifications of histones or nucleic acid, as therapeutic targets has been stimulated by the finding that their inhibitors exert profound effects, not only on survival, but also on the reproductive function of Schistosoma mansoni. Here, we review our current knowledge, and what we can infer, about the role of epigenetic mechanisms in schistosome development, differentiation and survival. We will consider which epigenetic actors can be targeted for drug discovery and what strategies can be employed to develop potent, selective inhibitors as drugs to cure schistosomiasis.

Fluorescence-Based Screening Assays for the NAD + -Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

Sirtuins are NAD+-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.